Tobacco mosaic virus (TMV) is an important tobacco disease, and the annual loss caused by it ranks forefront of the list of the top ten tobacco infectious diseases (Zhu Xianchao, 2002). There exist strain differentiations for TMV virus, such as TMV-Cg, TMV-U1 strain and the like, among which the TMV-U1 strain is the major strain of tobacco. Since the main cultivars of flue-cured tobacco such as K326, Yunyan 87 and the like are not resistant to TMV-U1 strain, generally, prevention and treatment measures such as cultivation of virus-free seedlings, chemical control and destruction of diseased plants in the field and the like are mainly taken. These measures have certain effects on controlling the occurrence and prevalence of TMV, but the cases of TMV prevail in a local field occur occasionally, leading to larger economic loss (Zhu Xianchao, 2002). Therefore, planting a TMV resistant variety is still the most fundamental and cost-effective mean for preventing and controlling TMV. The requirements for a disease-resistant variety include high resistance, no yield penalty and no agronomic traits disadvantages.
Currently, the TMV resistance resource of flue-cured tobacco is mainly from wild species of tobacco, Nicotiana glutinosa, the resistance of which is controlled by one dominant single gene (N). The N gene is resistant to TMV-U1 strain. N gene was cloned in 1994, which is the first NBS type of disease-resistant gene cloned in plants (Whitham, 1994). The length of the genome sequence of the N gene is 6,656 bp, including 5 exons and 4 introns, and belonging to a TIR-NBS-LRR type of disease-resistant genes. The N gene encodes a structure similar to the drosophila Toll protein and the extracellular domain of mammalian interleukin-1 receptor (Toll/interleukin-I receptor, TIR) on the N-terminal of the protein it encodes, and it also encodes a nucleotide-binding-site (NBS) and a leucine-rich repeat (LRR) domain (Whitham, 1994). The disease-resistant mechanism of N gene is that hypersensitive necrotic spots (necrotic spots) occur in the infected spots by virus, and the movement of TMV in a plant is limited by cell hypersensitive response caused by induction. After mediating the hypersensitive reaction, tobacco plants can obtain a systematic resistance, and produce a broad-spectrum resistance to the re-invasion of TMV or other similar pathogens (Whitham, 1994). By means of a series of conventional hybridization and backcrossing, the resistance of N gene is transferred from Nicotiana glutinosa into an oriental tobacco firstly, and then transferred into a flue-cured tobacco variety (Bagley, 2002). Transferring the resistance of N gene by using hybridization and breeding is actually transferring the chromosome segment carrying N gene (abbreviated as N introgression segment). Almost all the TMV resistant gene used by the breeding of TMV resistant flue-cured tobacco over the world is N gene. The representative varieties are TMV resistant flue-cured tobacco varieties Coker 176 and Speight H2O carrying N gene introgression segment, which are earlier commercially planted. Due to the linkage drags of lower yield, slower yellowing of the upper leaves, and so on, the flue-cured tobacco varieties carrying N gene could not meet the urgent need for leaf production. The linkage drags present in the chromosome segment derived from Nicotiana glutinosa which is closely linked to N gene, the narrow genetic background of the TMV resistant resources, which has been used for breeding, and the limitation of conventional breeding means, result in lacking breakthrough of TMV resistant flue-cured tobaccos breeding. Thus, the screening and identification of new TMV resistant gene in the tobacco germplasm resource bank is of great significance.
In addition, there is an N′ gene present in the wild tobacco varieties, and the corresponding avirulence gene is the coat protein (abbreviated as CP) gene of the virus of tobacco mosaic virus, belonging to CC-NBS-LRR type of disease-resistant genes (Sekine et al., 2012). N′ gene is resistant to TMV-Cg strain but not resistant to TMV-U1 strain.
For this, the present invention is intended to seek for a new gene resistant to TMV virus.